The long term goal of our research project is to elucidate the mechanism(s) that regulate alpha-fetoprotein (AFP) gene expression in liver during development and hepatocarcinogenesis. We will continue and extend our previous work in this application with an emphasis on DNA sequence-specific regulation of AFP gene expression. The project includes studies to localize and characterize the regulatory sequences of the AFP gene (including enhancer and silencer sequences) by transient expression assays with various AFP-CAT fusion gene constructs. After the cis-elements are clearly defined, the next goal will be to identify the regulatory sequence-binding proteins (trans-acting factors) in various AFP-producing and non-producing cells by gel mobility shift and DNase I- footprinting assays. The purification of trans-acting factors will be done by conventional column chromatography and DNA-affinity chromatography. The project will also include studies to characterize the purified trans-acting factors and to examine chemically and physically the interaction between DNA and proteins. An in vitro transcription system will be developed from hepatoma cells that will permit further characterization of transcription factors. The main objective of this application is not only to localize and characterize the regulatory sequences and transcription factors necessary for efficient (basal) transcription of the AFP gene, but also, importantly, to determine the cis- and trans-acting elements mediating the different actions of glucocorticoid hormones on AFP gene expression in hepatoma 7777 and McA-RH8994 cells.